Targeting of organs by immunoconjugates

ABSTRACT

Bioactive materials, e.g. therapeutic agents for treating a condition that afflicts a patient, are delivered to the afflicted organ in a site-specific manner by coupling the bioactive agent to an antibody or fragment or derivative thereof that recognizes a substance unique to that organ. For example, therapeutic agents for treating conditions of the prostate gland such as adenocarcinoma of the prostate, benign prostatic hypertrophy and prostatitis can be delivered to the prostate by coupling the thereapeutic agent to an antibody against a substance secreted by the prostate, e.g. prostatic specific antigen or prostatic acid phosphatase.

GOVERNMENT SUPPORT

[0001] The present invention was made with support from the Departmentof Veteran Affairs. The U.S. government may have certain rights in theinvention.

BACKGROUND OF THE INVENTION

[0002] Cancer, the second highest cause of death in the U.S.A., is oneof the most dreaded diseases. Unfortunately, the incidence of many typesof cancer (such as prostate, breast, ovary, colon, pancreas, lung) hasbeen increasing during the last 25 years. In addition, numerousinfectious diseases, many of which are drug resistant, are becomingprevalent in the industrialized world or are moving into theindustrialized world from the non-industrialized world. All solid organtumors (some are identified above) possess many clones or subpopulationsof cancer cells, which make them heterogeneous tumors that are verydifficult to treat. A common problem in developing therapies for each ofthese categories of diseases is targeting the therapeutic molecule(s) tomany clones of tumor cells that may be found in several loci inpatients.

[0003] Among cancers, adenocarcinoma of the prostate (CAP) is the mostprevalent neoplasm and is the leading cause of cancer death in males inthe U.S.A. In nearly 50% of patients, neoplastic prostate cells havespread at the time of diagnosis. Benign prostatic hypertrophy (BPH)results in over 400,000 surgical procedures in the U.S.A., which usuallyare done to correct urinary obstruction. This makes benign prostatichypertrophy the second most common reason for surgery in males in theU.S. Human prostate cancer, benign prostatic hypertrophy, andprostatitis require about 5 million physician visits, 900,000hospitalizations with 40,000 deaths, and costs over $3 billion/year inthe U.S.A. Similar estimates can be made for the industrializedcountries of Europe. In addition, the World Health Organization hasdetermined that BPH and prostatitis are world-wide health problems.Clearly, the current and potential need for a treatment for theseconditions is very large.

[0004] The current chemotherapeutic and endocrine treatments forprostate cancer as well as benign prostatic hyperplasia suffer from atleast two major limitations, lack of drug/treatment specificity and drugresistance. Lack of specificity has led to utilization of higher dosesof chemotherapeutic and endocrine drugs, which adversely affect manyorgans without tumors and have numerous unpleasant side effects inpatients. Furthermore, drug resistance (chemoresistance), usually due todevelopment of multidrug resistance proteins (Mdr) such asP-glycoproteins (Pgp), often negates the treatment effects in arelatively short time. Drug resistance varies from tumor to tumor and indifferent types of tumors. Most patients who develop chemoresistanttumors usually do not respond to other chemotherapeutic drugs or theircombinations. This results in treatment failure and death of thepatient, usually within a year. Because of the potential for drugresistance, the specific drug must act on many clones (orsubpopulations) of tumor cells directly and quickly to be effective,before they develop drug resistant proteins.

[0005] To overcome the limitations imposed by drug resistant proteins,the chemotherapeutic drugs must be highly specific to the target cellssuch as neoplastic cells of prostate, breast, ovary, colon, or othersolid organ tumors. Furthermore, the drug should have minimum to noeffects on the unrelated organs or tissues as well as minimal sideeffects in patients.

[0006] The existing treatments for prostate cancer include surgery,radiation, endocrine therapy, and chemotherapy. These often have limitedvalue, especially in patients with metastatic disease because thesetreatments are unable to specifically target metastatic cancer cells.Therefore, new approaches are required to improve treatment effects onprostate cancer as well as other solid organ neoplasms, such as those inthe breast, ovary, cervix, colon and lung. There is a need to targetbioactive compounds in other diseases and disorders as well.

[0007] Therefore, there is a need for highly specific drugs that couldovercome the above limitations and at the same time target manysubpopulations of neoplastic cells or dysfunctional tissues or organs.For cancers, it is important to target the tumors before they developdrug resistance for improvements in the treatments of prostate and othercancers.

SUMMARY OF THE INVENTION

[0008] The concept of targeting a specific organ requires considerationof biological characteristics of the normal organ as well as its tumorsand disease states. For example, in human prostate, prostatic specificantigen (PSA) and prostatic acid phosphatase (PACP) are organ-specificenzymes and are usually secreted by prostatic epithelial cells insufficient amounts. Therefore, the biological properties of theseenzymes can be used to develop highly specific drug conjugates thatwould then specifically target prostatic epithelial cells, but not othertypes of cells in other organs which usually do not secrete theseenzymes.

[0009] The present invention provides compositions and methods fordelivering bioactive compounds to target organ tissues. One embodimentof the method includes an immunoconjugate that recognizes a substance ina solid tissue. The immunoconjugate includes a polyclonal or monoclonalantibody, preferably a polyclonal antibody, as a binding or recognitionmoiety and a bioactive agent. The solid tissue can be a normal or benigntissue, preferably a tumor. In a specific embodiment, the tumor is aprostate tumor.

[0010] A polyclonal antibody has greater potential of recognizing manymore epitopes of the substance associated with the solid tissue, e.g.PSA or (PACP) in the case of the prostate, than a monoclonal antibody.Therefore, a polyclonal antibody would recognize more of the clones ortumor cells than a monoclonal antibody. Thus, an immunoconjugateprepared utilizing polyclonal antibody IgG or its fragments will bind tomost of the target cells that are producing an organ-specific enzyme,moiety, or substance.

[0011] The binding moiety is a molecule that recognizes and can bind toa molecule, substance and the like which is unique to the solid tissue.The binding moiety can be a polyclonal antibody or derivative orfragment thereof. Preferably, the binding moiety is an IgG. A conjugatein which the binding moiety is an antibody or antibody fragment that isconjugated to a bioactive substance, e.g. chemotherapeutic, hormonal orcytotoxic drugs, produces an immunoconjugate.

[0012] FIGS. 2-13 are micrographs resulting from in vitro experimentalprocedures discussed below.

[0013] FIGS. 14-18 are micrographs resulting from in vivo experimentswith nude mice discussed below.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

[0014] Delivery of an active substance to the site of its action isnecessary for the active substance to have its desired effect. One wayto deliver a bioactive substance or agent to a site where it hasactivity is to form a conjugate in which the bioactive agent is linkedto another molecule that has affinity for the organ to be treated. Thatis, a biologically active agent can be linked to a molecule that hasspecific affinity for the organ on which the bioactive substance is toexert its effect.

[0015] Such conjugates can take a variety of forms. A bioactive agentcan be linked to a small molecule that enters a cell or organ. Aconjugate can be formed between a bioactive agent and a protein orpeptide hormone that is recognized by a particular type of cell. Inaddition, there are numerous other types of proteins or polypeptidesthat can serve to deliver a bioactive agent to the desired organ,tissue, or cell. A preferred protein is an antibody that recognizes amolecule, a substance, or an epitope which is unique to a particularorgan. The antibody is preferably polyclonal, but monoclonal antibodiescould be used as well. The antibody can be an active portion of anantibody such as FAB or FC fragments. Conjugates between a bioactiveagent and an antibody are called immunoconjugates.

[0016] The immunoconjugates of the present invention target a desiredorgan using antibodies with affinity for a substance on the cell,tissue, or organ to carry bioactive substances to the cell. Antibodiescan be produced against substances found in human solid tissues andtumors of tissues such as prostate, breast, ovary, colon, pancreas andlung, and conjugated with bioactive agents to produce specificimmunoconjugates. The conjugates which include the bioactive agent canconcentrate the bioactive agent at the desired organ site and diminishunwanted side effects associated with non-specific drugs or bioactiveagents. Furthermore, the binding of bioactive agents to antibodymolecules can protect the bioactive agent from enzymatic degradation andprevent its rapid excretion. Selective delivery of the immunoconjugatesto the target sites can also lead to the use of smaller doses ofbioactive agents while maintaining effective treatment.

[0017] Immunoconjugates can be cytotoxic or can have other bioactivity.Typically, immunoconjugates have their effect inside the target celland/or plasma membranes and specific receptors on cell surfaces. Theimmunoconjugate can enter the cell by several mechanisms, such asendocytosis by the target cells, by diffusion, by facilitated diffusion,or by active carrier-mediated transport. Some conjugates enter thetarget cells by endocytosis and release their bioactive substance at thelysosomal membrane. Some immunoconjugates are not degraded at thesurface and enter the cells by endocytosis. The success ofimmunoconjugates at delivery of a bioactive substance can depend upontransport related factors such as the ability of the conjugate tolocalize and persist at the cell or tissue and its intracellular orintratissue penetration and metabolism.

[0018] The immunoconjugate can exert its effect without entering thecell as well. Internalization of the immunoconjugate may not benecessary since the conjugate may exert its bioactivity by, for example,action within the cell membrane. The immunoconjugate may bind tospecific receptors on the cell surface and elicit an inhibitory responsein the target cells, but not in non-target tissues. It may also beinternalized by endocytosis, diffusion, or active transport mechanism.

[0019] Another mechanism involves inhibition of DNA synthesis in thetarget cells because the immunoconjugate will reach the target cells viaantibody IgG. For example, a chemotherapeutic drug such as5′-fluorouracil or its derivatives inhibit thymidine synthetase(thymidine synthase) and thus, DNA synthesis is inhibited in targetcells. Without DNA synthesis, a neoplastic or any type of cell can notdivide or proliferate, and cell death will follow. In treatingconditions of the prostate, for example, with the specificity ofPSA-IgG-drug conjugate, the immunoconjugate would bind to prostatictumor cells and would inhibit DNA synthesis in the cell type of choice.Thus, the growth of the tumor is greatly decreased, and cell death willbe greatly enhanced. This mechanism will function in prostate cancer andbenign prostatic hyperplasia, including some breast and ovarian tumorsproducing PSA.

[0020] Immunoconjugates can show activity in a variety of metabolicdisorders, disease states, and physiological conditions. For example,conjugates can inhibit the growth of tumors of androgen-sensitive organssuch as testis, ventral prostate, and seminal vesicle, all of which haveandrogen-sensitivity. Immunoconjugates may also be useful to targetbioactive molecules to the locus of disease in conditions such asinfectious diseases including those caused by viruses or parasites, andmetabolic and inflammatory disorders of the organs, and the like.Immunoconjugates show activity against human disorders, such as prostatecancers or breast cancers, and in animal diseases and animal models ofhuman diseases. The present immunoconjugates are particularly desirablefor targeting solid organ cancers.

[0021] As one example, antibody IgGs to PSA or PACP proteins, whenconjugated to 5′-Fluoro-2′deoxyuridine (5′-FU-2′d) or otherchemotherapeutic agents, can form immunoconjugates that bindspecifically to PSA or PACP antigens present in prostate (neoplastic andnon-neoplastic) cells in the prostate gland. Since the immunoconjugatebinds preferentially to PSA or PACP antigens in the prostatic cells, thespecific immunoconjugate would readily reach the target cells andinhibit DNA synthesis and elicit cell death and/or reduce, or evenabolish, cell proliferation much before the prostatic cells are able todevelop multidrug resistant proteins. Non-tumor conditions such asprostatitis also can be treated in this manner.

[0022] Some breast and ovarian tumors have been shown to produceprostatic specific antigen, but not prostatic acid phosphatase.Therefore, in PSA-producing tumors, PSA-IgG-drug conjugate could be usedto target neoplastic cells in the organs, much like prostatic tumors. Inthese tumors, the immunoconjugate would also inhibit thymidine synthaseand thus, DNA synthesis. With the inhibited DNA synthesis, the tumorwill cease to grow or proliferate, and cell death would occur.

[0023] Antibodies can be raised to a variety of substances, haptens,proteins, or antigens. As used herein, antigen refers to a substance ormolecule recognized or bound by an antibody. The distribution of theantigen through the organism or cell determines where in the organismthe antibody will bind and be localized or delivered. An antigen that isdistributed throughout an organism or body would localize the antibodyor immunoconjugate throughout that organism or body. Conversely, anantigen that is found only on a particular cell type in a particularorgan would localize the antibody or immunoconjugate to that specificcell population in that specific organ. Furthermore, some antigens areexpressed only in dysfunctional or infected cells or organs, whichallows them to be used to target antibodies or immunoconjugates to thatparticular cell type or organ.

[0024] For example, as noted above, PSA and PACP are antigens which areuniquely produced by the prostate gland in humans. As a further example,in mammals the mammary gland uniquely amongst all organs producescasein. Casein would therefore be a suitable molecule or substance touse to localize an immunoconjugate to the mammary gland. Furthermore,casein is readily available as a reagent to use to raise antibodies fromnumerous sources. Polyclonal (or monoclonal) antibodies raised againstcasein can then be used to form immunoconjugates.

[0025] In addition, the human mammary gland secretes caseins (alpha,27,300 daltons and beta, 24,000 daltons) during lactation, but smallamounts are also elaborated in differentiated mammary glands ofnon-pregnant, adult women during the reproductive cycle. Caseins areusually part of mucins that are sometimes called milk mucins. There areat least 5 types of mucins, of which MUC 1 is well characterized, MUC 2to 5 less so (Sandra et al., In: Breast Epithelial Antigens, R. L.Ceriani, editor, Plenum, New York, 1991). MUC 1 is the only human mucinfor which a full length cDNA has been described. Many different types ofantibodies against mucins have been produced around the world, and itappears that for breast cancer, more than 90% of them react with mammarymucins (Xing and McKenzie In: Breast Epithelial Antigens, R. L. Ceriani,editor, Plenum, New York, 1991; Cancer Cells, 2:75-78, 1990). Mucins aretransmembrane molecules and can be demonstrated by polyclonal antiserum(CT-1). The antiserum reacts with MUC 1 and is available from commercialsources. Antibodies against caseins or mucins could also be produced inmany research laboratories using the methods described below.

[0026] The human gastric epithelium and its neoplasms also producemucins (Ho et al., Cancer Res., 55:2681-2690, 1995; Ho and Kim, CancerBiol., 2:389-400, 1991; Ho et al., Cancer Res., 53:641-651, 1993) andthus this is another the formation of metastatic tumors elsewhere in thebody. Sinha et al. (see above) showed that cathepsin B, a cysteineprotease, is involved in the breakdown of basement membranes in theangiogenic blood vessels of human prostate cancer. Therefore, endogenousinhibitors (such as cystatin A, B, and/or C) of cathepsin B could beused to inhibit degradative roles of cathepsin B at the sites ofangiogenesis or tumor invasion. For example, cystatin A could beconjugated to PSA-antibody IgG using the earlier described methods andthen the PSA-IgG-cystatin A immunoconjugate could be used to inhibitformation of new blood vessels at the tumor sites. Since cathepsin B isalso involved in tumor invasion, PSA-IgG-cystatin A or B would alsoinhibit tumor cell invasion of the stroma. Without angiogenesis, growthof prostate tumor would not occur. Likewise, PACP-IgG could be used forconjugating cystatin A, B or C.

[0027] Angiogenesis is essential for tumor growth (Sinha et al., citedabove) as well as the growth of normal tissues during wound healing andother repairs. A variety of chemicals, agents, and/or inhibitors (suchas Thrombospondin: Koch et al., Pathobiol., 61:1-6, 1993; Heparin:Norby, Haemostasis 23 (Suppl 1):141-149, 1993; Heparin adipic hydrazidewith/without cortisol: Thorpe et al, Cancer Res., 53:3000-3007, 1993;medoxyprogesterone: Folkman, Adv. Cancer Res., 43:175-202, 1985;Klagsburn and D'Amore, Ann. Rev. Physiol., 53:217-239, 1991) have beenproposed to inhibit angiogenesis. Most of these agents have not beensuccessful because they could not be delivered to the specific sites ofnew blood vessel formation near the tumors. It is obvious thatinhibition of blood vessel formation throughout the human body will havenumerous detrimental effects. Using the present immunoconjugationmethods, the above inhibitors/molecules could be conjugated and thus,the newly formed immunoconjugate will be delivered to the target sitesto inhibit formation of new blood vessels which are often very closelyassociated with the prostatic glandular epithelium. In other words, avariety of angiogenic inhibitors can be conjugated with PSA and/orPACP-IgGs and delivered to the angiogenic sites where they would induceinhibition of angiogenesis. Delivery of angiogenic inhibitors viaPSA-IgG should inhibit tumor growth at the early stages as well as tumorgrowth and tumor cell invasion in the stroma.

[0028] The roles of receptors (such as androgen, estrogen, orprogesterone) in protein synthesis have been defined for human andanimal organs (Luke and Coffey, J. Androl., 15:41-51, 1994; DeWinter etal., Am. J. Pathol., 144:735-746, 1994; Srinivasan et al., Micros. Res.& Techniques, 30:393-304, 1995; Khan et al., Cancer Res., 54:993-997,1994; O'Malley and Tsai, Biol. Reprod., 46:163-167, 1992). The targetorgan cells will not proliferate in the absence of protein synthesis.For example, androgen receptors are found in a variety of organs andtissues (such as prostate, genital skin, testis). Any immunoconjugateshould not target normal organs. In contrast, PSA occurs predominantlyin the prostate gland. This situation can be remedied by using PSA-IgG(or PACP-IgG), which are organ-specific markers. Using PSA/PACP-IgG, asa carrier protein, much as it has been used for 5′-Fu and itsderivatives, the anti-receptor antibody IgGs can be delivered to thetarget organ tumors. In other words, PSA-IgG will function as a carriermolecule for anti-androgen receptor IgG to the target prostateepithelial cells and their tumors. The PSA-IgG-anti-androgen receptorIgG will then inhibit protein synthesis in prostatic cells. Therefore,when PSA-IgG is conjugated to anti-androgen receptors, PSA-IgG willdeliver anti-androgen receptor antibody IgG only to prostate cells, bothepithelial and stromal, and not to testis or genital skin which do notpossess prostatic specific antigen. This property makes this doubleimmunoconjugate very specific for inhibiting protein synthesis inprostatic tumors. Likewise, when PSA-IgG is conjugated to anti-estrogenand/or progesterone receptors, these receptors will be carried to aspecific organ (some breast and ovarian tumors produce PSA) via PSA-IgG.In turn, this will inhibit protein synthesis in breast-ovarian tissuesand tumor growth will be inhibited.

[0029] In summary, with respect to prostate treatment in accordance withthe present invention, PSA-IgG (or PACP-IgG) functions as a carriermolecule to which a variety of molecules such as chemotherapeuticdrugs/toxins, inhibitors to angiogenesis, enzymes, and even antibodyagainst steroid receptors can be conjugated and delivered in a specificmanner to the target tissues. Multiply-coupled agents also are possible.For example, a PSA-IgG-anti-androgen receptor-IgG-drug combination mightbe used. The present invention can be applied to other organs usingantibodies to substances found in those organs.

[0030] Production of monoclonal and polyclonal antibodies against avariety of antigens, peptide and other agents/molecules has becomeestablished in research and commercial laboratories (ImmunochemicalMethods by R. J. Mayers and J. H. Walker, editors, Academic Press, 1987;Practical Immunology, L. Hudson and F. C. Hay, editors, Blackwell Sci.Publ., 3rd edition, 1989; Antibody Techniques by V. S. Malik and E. P.Lillehoj, editors, Acad. Press, 1994; Monoclonal Antibodies: Principlesand applications, J. R. Birch and E. S. Lennox, editors, Wiley-Liss,1995). The following protocol can be used to produce polyclonal antibodyagainst PSA as well as PACP. Prostatic specific antigen or prostaticacid phosphatase molecules will be isolated from semen of normal healthymales and purified of contaminating enzymes, proteins, and othersubstances (see for details, Practical Immunology, L. Hudson and F. C.Hay, editors, Blackwell Sci. Publ., 3rd edition, 1989; AntibodyTechniques by V. S. Malik and E. P. Lillehoj, editors, Acad. Press,1994). The affinity purified enzymes/antigens will be suspended inphosphate buffer saline NaCl (0.5 ml) complete with Freund's adjuvant(0.5 ml). The suspension is then injected intramuscularly at multiplesites in male New Zealand rabbits. Two weeks after the initial does, abooster injection is given intramuscularly. Booster injections arerepeated up to four times at biweekly intervals. Pre-immune serum iscollected before injecting the antigen/enzymes. After immunizations,serum is collected again and purified for the immunoglobulin G (IgG)fraction, i.e., traces of contaminating antibodies and otherimmunoglobulins will be removed by solid-phase absorption techniques.The antibody will also be purified against PSA/PACP affinity columns.The specificity and purity of PSA-IgG can be established byradioimmunoassay and chemiluminescence immunoassay techniques (seePractical Immunology, L. Hudson and F. C. Hay, editors, Blackwell Sci.Publ., 3rd edition, 1989; Antibody Techniques by V. S. Malik and E. P.Lillehoj, editors, Acad. Press, 1994). Polyclonal antibodies againstPACP and mucins (including casein alpha or beta) could also be preparedusing the above cited literature. In fact, most of these antibodies arereadily available from commercial sources and their production could becustomized for specific needs at a very reasonable cost. In summary,production of both monoclonal and polyclonal antibodies against selectedmolecules identified in this application is not a limitation forpreparing immunoconjugates.

[0031] Polyclonal antibody against PSA is most suitable because itrecognizes nearly all epitopes of prostatic specific antigen in humanprostate whereas monoclonal antibody will recognize a single and similarepitopes of PSA. For some prostate cancer cases, a cocktail ofmonoclonal antibody IgGs could be made and then the IgGs would beconjugated to chemotherapeutic drugs or toxins. In well-differentiatedtumors, especially when prepared for a specific patient, whethermonospecific polyclonal or monoclonal antibody against PSA, the latterIgGs would be conjugated. Much as the polyclonalIgG-drug-immunoconjugates, monospecific or monoclonal antibody IgGswould be conjugated and the later immunoconjugates could prove to beeffective. Likewise, monospecific/monoclonal PACP antibody IgGs will beconjugated with chemotherapeutic drugs and toxins/inhibitors.

[0032] Monoclonal or polyclonal antibodies against PSA and PACP can beproduced using a variety of techniques (see Practical Immunology, L.Hudson and F. C. Hay, editors, Blackwell Sci. Publ., 3rd edition, 1989;Antibody Techniques by V. S. Malik and E. P. Lillehoj, editors, Acad.Press, 1994). Polyclonal antibody could be produced in rabbit usingmonospecific PSA antigen, much as it was done for cathepsin B (Sinha etal., Prostate, 26: 171-178, 1995). Alternatively, a cocktail ofmonoclonal PSA antibody will be produced using mice, rat, or hybridomatechnology (Antibody Techniques by V. S. Malik and E. P. Lillehoj,editors, Acad. Press, 1994) or two or more monoclonal antibodies will bemixed together to get a cocktail of antibody IgGs. Regardless, the IgGmolecules, whether derived from polyclonal and monospecific ormonoclonal antibody methods of preparation, would function as carrierproteins for conjugating a variety of agents/drugs. PSA-IgG moleculewould deliver the conjugated drug/toxins/other agents to human prostatictumors. In the event there is immunologic reaction against a givenimmunoconjugate, immunosuppressive drugs could be used. However, thereshould not be any immunologic reactions before 14 to 18 days after theinitiation of treatment with an immunoconjugate and this ought to beestablished for a given immunoconjugate.

[0033] The present immunoconjugates can include a variety of bioactiveagents that exert a variety of effects. Some bioactive agents are sopotent that exposure of the cell to a single molecule of the bioactiveagent is sufficient to have the desired biological effect, such askilling the cell. But bioactive agents can have varying degrees ofpotency and can work by many mechanisms. Mechanisms for bioactive agentsor toxins include inactivating DNA and protein synthesis or recognizingspecific proteins or protein complexes in the cell and interfering withtheir action. Bioactive agents can be enzyme inhibitors, receptorantagonists, molecules that interfere with macromolecular interactions,antimetabolites, and the like.

[0034] The bioactive agent can be a chemotherapeutic agent such asmethotrexate, doxorubicin, adriamycin, cis-platinum, tamoxifen,estrogen, progesterone, mitomycin C, 5′-fluorouracil, or5′-fluoro-2′-dioxyuridine, vinblastin, doxorubicin and the like.Bioactive agents such as chemotherapeutic agents or anti-metabolites canexert bioactivities such as inhibiting DNA synthesis, either directly orindirectly, in tumors and cancers such as those of the breast, colon,and ovary. The bioactive agent can be a drug such as daunomycin orouabain. The bioactive agent can be a radio-labeled compound or aradionuclide such as I¹²⁵ or I¹³¹ and the like. The bioactive agent canbe a plant toxin or a bacterial toxin such as ricin, abrin, diphtheriatoxin, or cholera toxin.

[0035] Patient treatment using the method of the present inventioninvolves administering therapeutic amounts of the immunoconjugatecomposition. In the context of the present invention, the terms “treat”and “therapy” and the like refer to alleviate, slow the progression,prophylaxis, attenuation or cure of existing disease. An immunoconjugatecomposition may be formulated with conventional pharmaceuticallyacceptable vehicles for administration by injection. These vehiclescomprise substances which are essentially non-toxic and non-therapeuticsuch as water, saline, Ringer's solution, dextrose solution, Hank'ssolution, or the like. It is to be understood that immunoconjugateformulations may also include small amounts of adjuvants such as buffersand preservatives to maintain isotonicity, physiological and pHstability. Preferably, the immunoconjugate is formulated in purifiedform substantially free of aggregates and other protein 0.001 to about10 mg/ml and preferably from about 0.01 to about 1.0 mg/ml. Theconcentration may vary depending upon conjugate and concentration of IgGand drug molecules.

[0036] The dose of the immunoconjugate formulation to be administeredwill depend upon the patient and the patient's medical history, and theseverity of the disease process. However, the dose should be sufficientto suppress the disease state, for example to slow or stop theprogression of prostate cancer. Dosages for adult humans envisioned bythe present invention and considered to be therapeutically effectivewill range from about 0.001 to about 10 mg/subject/day and preferablyfrom about 0.01 to about 2.0 mg/subject/day; however, lower and higheramounts may be more appropriate. In addition, to the extent that thepresent immunoconjugate includes a bioactive agent that is currentlyused for treating the condition in question, current dosage regimens canbe taken into consideration, it being noted that the administration ofthe bioactive agent in the form of the immunoconjugate in the presentinvention should allow the administration of lower levels of thebioactive agent to achieve the desired beneficial effect.

[0037] The immunoconjugates can be delivered directly to the organ to betreated. They also can be administered directly to the blood stream,e.g. by intravenous injection or intramuscular administration. Oraladministration may be possible in some cases. The immunoconjugatesshould be formulated with appropriate carriers depending upon theintended mode of administration.

[0038] The invention will be further described by reference to thefollowing examples, which should be considered to be non-limiting.

Methods for Producing Antibodies to Substances in Solid Tissues

[0039] Production of monoclonal and polyclonal antibodies against avariety of antigens, peptide, and other agents/molecules has becomeestablished in research and commercial laboratories and has beenextensively reported in the literature (Immunochemical Methods by R. J.Mayers and J. H. Walker, editors, Academic Press, 1987; PracticalImmunology, L. Hudson and F. C. Hay, editors, Blackwell Sci. Publ., 3rdedition, 1989; Antibody Techniques by V. S. Malik and E. P. Lillehoj,editors, Acad. Press, 1994; Monoclonal Antibodies: Principles andapplications, J. R. Birch and E. S. Lennox, editors, Wiley-Liss, 1995).The following protocol can be used to produce polyclonal antibodyagainst PSA as well as PACP. Prostatic specific antigen or prostaticacid phosphatase molecules will be isolated from semen of normal healthymales and purified of contaminating enzymes, proteins and othersubstances (see for details, Practical Immunology, L. Hudson and F. C.Hay, editors, Blackwell Sci. Publ., 3rd edition, 9189; AntibodyTechniques by V. S. Malik and E. P. Lillehoj, editors, Acad. Press,1994). The affinity purified enzymes/antigens will be suspended inphosphate saline NaCl (0.5 ml) complete with Freund's adjuvant (0.5 ml).The suspension is then injected intramuscularly at multiple sites inmale New Zealand rabbits. Two weeks after the initial dose, a boosterinjection is given intramuscularly. Booster injections are repeated upto four times at biweekly intervals. Pre-immune serum is collectedbefore injecting antigen/enzyme. After immunizations, serum is collectedagain and purified for the immunoglobulin G (IgG) fraction, i.e., tracesof contaminating antibodies and other immunoglobulins are removed bysolid-phase absorption techniques. The antibody is then purified againstPSA/PACP affinity columns. The specificity and purity of PSA-IgG shouldbe established by radioimmunoassay and chemiluminescence immunoassaytechniques (see Practical Immunology, L. Hudson and F. C. Hay, editors,Blackwell Sci. Publ., 3rd edition, 1989; Antibody Techniques by V. S.Malik and E. P. Lillehoj, editors, Acad. Press, 1994). Polyclonalantibodies against PACP and mucins (including casein alpha or beta)could be prepared in a similar manner. In fact, most of these antibodiesare readily available from commercial sources and their production couldbe customized to specific needs at a very reasonable cost. In addition,there are companies (such as American Biogenetic Sciences, Inc. NewYork, N.Y.) who market humanized antibodies for injections and treatmentin patients. The humanized PSA and PAcP antibodies then would beconjugated with the selected drugs, toxins or other bioactive agent.Humanization of the antibody will greatly reduce or abolish adverseeffects of foreign proteins in patients. Immunoconjugates to be testedin humans or animals should be made with sterile, pyrogen-free solution,and prepared in pharmaceutically acceptable, buffered, isotonic aqueoussolution. Exotoxins and endotoxins should be removed before injectionsin humans and animals. All of these techniques are familiar to thoseskilled in this art and thus are not discussed in detail.

Methods for the Conjugation of an Antibody to a Bioactive Substance

[0040] There are a variety of methods to conjugate bioactive substancesto antibodies, or other targeting proteins, to form immunoconjugates.The bioactive substance can first be conjugated to a carrier protein,such as human serum albumin, and this complex is then chemically linkedto an antibody. Alternatively, the bioactive substance can be linked tothe antibody, or other targeting protein, either directly or through asmall organic or inorganic linker. Examples of such linkers includecis-aconitase and other linkers known in the art.

[0041] Another linker is Sulpho-SMPB Succinimidyl-4-(p-maleimidophenyl)butyrate (Pierce Chem. Co., Rockford, Ill.). SMPB aromatic crosslinkershave been shown to improve the yield of immunotoxin conjugates (Myers etal. J. Immuno. Methods 121, 129-142, 1989; Pietersz, G.A. BioconjugateChem. 1, 89-95, 1990; Cumber et al., Methods Enzymol. 112, 207-225,1985; Akerblom et al., Bioconjugate Chem. 4, 455466, 1993).

[0042] Several methods have been used to conjugate PSA-IgG with5′-FU-2′d. For example, rabbit anti-human PSA antigen (Dako) wasconjugated with 5′-fluoro-2′-deoxyuridine 5′-p-aminophenylphosphate(Sigma, St. Louis, Mo.).

[0043] Conjugation of 5′-Fu-2′-d or 5′-FU-2′-d-5′-mp with PSA-IgG wasaccomplished using known conjugation and purification techniques (Myerset al., J. Immuno. Methods 121, 129-142, 1989; Pietersz, G. A.Bioconjugate Chem. 1, 89-95, 1990; Cumber et al., Methods Enzymol. 112,207-225, 1985; Akerblom et al., Bioconjugate Chem. 4, 455-466, 1993;Whiteley et al., Biochem. 13, 2044-2050, 1974). Briefly,5′-Fu-2′-d-5′-mp was dissolved in 20 μl dimethylformamide (Myers et al.,J. Immuno. Methods 121, 129-142, 1989) and 200 μl 100 mM PBS (pH 7.8)was added. A 5 mole excess of Sulpho-SMPBSuccinimidyl-4-(p-maleimidophenyl) butyrate was dissolved in 100 mMphosphate buffer saline (PBS, pH 7.8) and then added to the abovemixture. The later mixture was incubated at 25° C. for 30 minutes, afterwhich the reaction was quenched with 2 mg bovine serum albumin (BSA) (10mg/ml) in 100 mM PBS (pH 7.8) for 30 minutes at 25° C. The mixture wasplaced over a Pharmacia PD-10 column equilibrated with 100 mM PBS (pH7.8). Column fractions were analyzed at 260 and 280 nm. The extent ofconjugation was determined using UV Spectrophometer. The unconjugatedrhodamine and fluorescein were removed by gel column chromatography(Whiteley et al., Biochem. 13, 2044-2050, 1974). Using methods ofWhiteley et al. (Biochem. 13, 2044-2050, 1975), the purity of thePSA-IgG-drug was established. The Drug-Immunoconjugate was reactive toPSA by indirect immunoassay (Albrecht et al., Clin. Chem. 40, 1970-1971,1994). Conjugation was also determined using UV spectrophotometry andimmunoreactivity of PSA by chemiluminescence assay.

[0044] Similar methods have been used to conjugate PACP-IgG with5′-fluoro-2′-deoxyuridine. In this specific conjugate, approximately 67molecules of 5′-Fu-2′-d-5′-mp were conjugated/molecule of IgG.Chemotherapeutic drugs, rhodamine, and FITC were obtained from SigmaChem. Co. (St. Louis, Mo.). Likewise, 5′-fluoro-2′-deoxyuridine and5′-Fu2′-d-5′-mp were conjugated with or without rhodamine orfluorescein. Drugs also were conjugated with FITC alone for control testpurposes using similar procedures.

Human Prostate Tissue Collection

[0045] Human prostate tissue can be obtained from patients who undergoprostatectomy, cystoprostatectomy, or transurethral resection of theprostate (TURP). Prostatectomy samples are preferably collected frompatients who have not been treated with cytotoxic or endocrine therapybefore surgery. Data about the patients including age, grade and stageof the disease, and serum PSA values can be obtained and correlatedbefore and after immunoconjugate treatment.

[0046] Samples can be dissected immediately after surgery for study. Allprostate specimens should be collected aseptically and will includespecimens representing tumors with a Gleason histologic scores of 2 to10 (Human Pathol. 23:273-279, 1992). Benign prostatic hypertrophysamples can be obtained from patients undergoing prostatectomy or TURPfor benign prostatic hypertrophy as established by histology. ‘Normal’prostate samples can be collected from cystatectomy patients and can beused as control tissues.

[0047] Portions of prostate samples are then evaluated by histology toestablish definitive diagnosis of either benign prostatic hypertrophy orcancer including the grades or histologic scores or both. Portions ofcolon, kidney, bladder and lung tissues will be collected to establishthe specificity of immunoconjugates along with the negative and positivecontrols.

Preparation of Tissue or Organ Specimens

[0048] Prostate tumor and control prostate samples are dissected andprocessed according to protocols known in the art, then frozen in liquidnitrogen, or fixed in 10% buffered formalin and/or 3 to 4% bufferedparaformaldehyde. In general, samples range from about 200 mg to 2g.Excess tissue frozen in liquid nitrogen can be stored at −70° C. forfuture biochemical studies or fixed in formalin as needed. Randomlyselected pieces can be used in organ culture and immunoconjugatetreatments. Prostate and control tissues can be evaluated before andafter organ culture and before and after drug treatments.

[0049] The procedures for sample preparation, fixation, embedding,sectioning, immunocytochemistry, in situ hybridization, andimmunoelectron microscopy techniques are known in the art and will notbe described in detail. Briefly, fixed and unfixed specimens can beembedded in OCT (Lab Tek Products, Naperville, Ill.), sectioned at 4 to6 μm with a cytostat at −20° C., placed on specially cleanedpoly-L-lysine coated slides and air dried in cold air. The remainingfixed specimens can be washed in 0.5 M NaCl for 10 minutes and processedfor paraffin embedding. In general, pathological diagnosis is determinedfrom cryostat or paraffin sections. Each specimen can be gradedaccording to the Gleason grading system. (Human Pathol. 23: 273-279,1992).

[0050] Prostate, colon, kidney, and lung samples can be evaluatedhistologically before and after short-term organ culture. Samples can betreated with PSA and PACP-IgG-immunoconjugates to establish optimum timeand dilutions. PACP is an enzyme made predominantly by the prostategland (Sakai et al., J. Urol. 149, 1020-1023, 1993; Sinha et al.,Prostate 13, 1-15, 1988). A study was undertaken to demonstrate thathuman prostatic acid phosphatase (PACP) is also specific for prostaticepithelial glands and stromal tissues. Immunoconjugate effects can beevaluated in treated samples and compared with the untreated samplesafter localization of one or more markers. Markers include those forthymidine synthase, cell proliferation (PCNA), cell death (DNAfragments), and P-glycoprotein as well as unconjugated PSA and PACPantibodies. Negative controls can include normal rabbit serum. Suchtesting can establish immunoconjugate localization patterns andphenotypic characteristics of prostatic cells at light and electronmicroscopic levels.

Localization of Thymidine Synthase

[0051] Human prostate tissues collected and prepared as described abovewill be used to demonstrate localization pattern of thymidine synthasein immunoconjugate-treated and untreated samples. Since 5′-Fu and itsderivatives inhibit thymidine synthase, immunoconjugate-treated sampleswill show a markedly reduced thymidine synthase localization pattern inthe affected cells than the untreated samples. Differential analysiswill reveal the effects of immunoconjugate on the target cell DNAsynthesis thereby decreased cell proliferation and increased cell death.Several studies have shown effects of 5′-Fu and its derivatives on humantissues. (Goerlach et al, Bioconjugate Chem. 2, 96-101, 1991; Krauer etal., Cancer Res. 32, 132-137, 1992; Uchida et al., Anticancer Res. 10,779-784, 1990; Johnson et al., Cancer Res. 55, 1407-1412, 1995; Liu &Santi; Biochim. Biophys. Acta 1209, 89-94, 1994). These drugs inhibitthymidylate synthetase in a variety of cell lines (Goerlach et al.,Bioconjugate Chem. 2, 96-101, 1991; Krauer et al., Cancer Res. 32,132-137, 1992; Uchida et al., Anticancer Res. 10, 779-784, 1990; Johnsonet al., Cancer Res. 55, 1407-1412, 1995; Liu & Santi; Biochim. Biophys.Acta 1209, 89-94, 1994; Cobb, Cancer Immunol. Immunother., 28: 235-240,1989).

Localization of Proliferation Cell Nuclear Antigen (PCNA)

[0052] Human prostate tissues were collected and prepared as describedin Examples 2 and 3 above. These tissue specimens were exposed toantibodies that recognize PCNA and/or KI 67 both are used as markers ofcell proliferation. Binding of these antibodies to the tissue wasvisualized by light microscopy. Results can be compared betweenconjugate-treated and untreated samples for drug induced inhibition ofcell proliferation. Proliferating cells can be quantitated for objectiveanalysis.

Localization of Fragmented DNA

[0053] The patterns of cell death, before and after immunoconjugatetreatments of human prostate, can be established by localization of DNAfragments in apoptotic cells by the methods of Wijsman et al. (J.Histochem. Cytochlem. 41:7-12, 1993) and Landstrom et al. (Cancer Res.,54:4281-4284, 1994).

[0054] Briefly, prostate pieces treated with immunoconjugate anduntreated pieces can be fixed in 3 to 4% paraformaldehyde, embedded inparaffin or paraplast, sectioned at 4 to 6 μm, and mounted onpoly-L-lysine coated slides. Sections will be deparaffinized,rehydrated, and digested with pepsin (0.5% pepsin) in HCl (pH 2) at 37°C. Digestion is stopped by washing in running water and then in buffer A(50 mM Tris-HCl, 5 mM MgCl₂, 10 mM B-mercaptoethanol, and 0.005% bovineserum albumin (Fraction V; Sigma Chemical Co., St. Louis, Mo.; pH 7.5)for 5 minutes. The tissue sample is then incubated at 15° C. with bufferA containing 0.01 mM dATP, dCTP, and dGTP (Boehringer-Mannheim,Indianapolis, Ind.) and 0.01 mM biotin-11-dUTP and 4 units/ml DNApolymerase 1 (Sigma). After blocking endogenous peroxidase in PBScontaining 0.1% H₂O₂, sections are washed twice in PBS and incubatedwith horseradish peroxidase-conjugated avidin (Vector Labs., Burlingame,CA) diluted in 1:100 in PBS containing 1% BSA and 0.5% Tween 20 for 30minutes at room temperature before developing with diaminobenzdine(Sigma). For negative controls, DNA polymerase is excluded in thenucleotide mixture.

Localization of P-glycoprotein

[0055] Analysis of immunohistochemical localization of monoclonalantibody C219 (Signet Lab., Dedham, Mass.) in prostate tissue samplesallows estimation of the presence or absence of the drug resistantproteins (P-glycoprotein) in untreated and immunoconjugated-treatedprostate cells. C219 recognizes an internal, highly conserved amino acidsequence found in both Mdr-1 and Mdr-3 isoforms of P-glycoproteins,which are found on a variety of tumors and cell lines. Localizationtechniques that determine the effect of dilution of the antibody inhuman prostate are known. For positive control, kidney tissues, drugsensitive SKOV3 cell line, and a variant of the cell line which is drugresistant can be used. The cells can be obtained from the American TypeCulture Collection (Rockville, Md.).

[0056] Techniques for Localization of Two or More Antigens Some studiesmay require localization of two or more antigens or markers in the samesections. For example, the present methods can localize laminin byimmunoperoxidase (IP) and cathepsin B by alkaline phosphatase (AP) inhuman prostate sections. The sequence of localization can be critical.For example, when laminin is localized by immunoperoxidase first andthen cathepsin B by AP, suitable reaction products for both antigens areobtained. However, when the reverse order was employed, the alkalinephosphatase reaction products for cathepsin B were washed out.

[0057] Using these techniques, the distributions of immunoconjugatereaction products in relation to the localization of antibodies tothymidine synthase, PCNA, P-glycoprotein, DNA fragments, and othermarkers of a disease state or metabolic disorder can be defined.

Immunoconjugate Treatment of Prostate Cancer Tissues

[0058] Antibody PSA IgG was conjugated with 5′-FU-2′d or5′FU-2′d-biotin. The PSA-immunoconjugate is suitable for studying humanprostate cancer, benign prostatic hypertrophy, and prostate cell lines.The data shows that the immunoconjugate localized in glandularepithelia, but not in the stromal cells (FIGS. 2 and 3 discussed indetail below). Since PSA is secreted by epithelial cells, our studyindicates that the PSA-immunoconjugate is specific to the prostateglands. This is notable because both benign prostatic hypertrophy andneoplastic tumors are of epithelial cell origin. These studies indicatethat there should not be any problem in identifying the sites ofimmunoconjugate localization labeled with markers in untreated andtreated human prostate and prostate cell lines.

Immunoconjugate Treatment of Prostate Cell Lines

[0059] The objective is to determine effects of PSA and PACPimmunoconjugates (at 0, 1, 24, 48, 72 hours) on DU-145 and LN-CAPprostate cell lines and to establish patterns of thymidine synthaseinhibition, reduced/decreased DNA synthesis, cell proliferation, celldeath, and P-glycoprotein localizations. By comparing results betweenuntreated and treated samples, one can establish the effects ofimmunoconjugates on these cell lines.

[0060] Conjugated drugs will reach the target sites because of thebinding ability of the PSA-IgG antibody to PSA antigens which areusually found in prostatic epithelial cells. 5′-Fu-2′-d or5-FU-2′-d-5′-mp, when conjugated with PSA-IgG, could be localized in thesame group of prostatic cells. Using conjugation and purificationtechniques (Whiteley et al., Biochem. 13:2044-2050, 1974), PSA-IgG wasconjugated with rhodamine and 5′-Fu-2′-d-5′-mp with fluorescein and thenconjugated together. SMPB aromatic crosslinkers were used to improve theyield of immunotoxin conjugates. The extent of conjugation wasdetermined by UV spectrophotometry. Immunoreactivity of the conjugatewas established by chemiluminescence assay. Approximately, 81 moleculesof 5′-Fu-2′-d-5′-mp were conjugated/molecule of IgG. Rhodamine Bisothiocyanate and Fluorescein isothiocyanate were purchased from Sigma.

[0061]FIGS. 3, 4, 5, 6, 11 and 12 illustrate the localization of bothPSA and 5′-Fu-2′-d-5′-mp in the same group of cells. In contrast, thedrug conjugated with fluorescein alone did not localize in epithelialcells (FIG. 9). These are discussed in more detail below.

[0062] Thymidylate synthase is the target enzyme for 5′-fluorouracil andits derivatives. The enzyme (EC 2.1.1.45) is involved in DNA synthesis.It is known that both 5′-Fu2′-d-5′-mp and5′-fluoro-2′-deoxycytidine-5′-monophosphate inhibit thymidylatesynthase. Inhibition of DNA synthesis should result in decreased cellproliferation, increased cell death and increased synthesis of multidrugresistant proteins in prostate samples and prostate cell lines.

Animal Models of Human Prostate Cancer

[0063] Animal prostates do not express prostatic specific antigens orhuman prostatic acid phosphatase. However, human prostate pieces cangrow when implanted in nude mice although they do not metastasize. TheDU-145 human prostate cell line derived from a human prostatemetastasized to brain does not metastasize in nude mice. The LN-CAP cellline can metastasize in adjacent tissues, if implanted with Matrigel innude mice, but the pattern of metastasis is not similar to metastasis ofhuman prostate cancer which metastasizes to bones, lymph nodes, brain,lungs and liver.

[0064] Human prostate cell lines PC-3, a derivative of the cell line1-LN, and DU-145, can grow in nude mice. These cell lines do not expressPSA antigens in cell culture or when grown in nude mice. ThePSA-immunoconjugates did not recognize these cells lacking PSA antigens.This indicates that PSA-immunoconjugates are specific for cellscontaining PSA antigens and not for cells without the antigens.

[0065] Human prostate tumor pieces were transplanted into nude miceprostates. Human prostate tumor pieces were allowed to grow in nude micefor over a month and then the mice were sacrificed. Histological and PSAlocalization showed that human prostate tumor grew in a nude mouseprostate and the tumor cells localized PSA indicating that human tissuecontinues to express PSA in the animal model. This demonstrates that thePSA-immunoconjugate is specific for human prostate tumors with PSAantigens even after growing in nude mice. Human prostate pieces wereimplanted surgically under the ventral skin and inside the ventralprostate of nude mice. Mice were kept alive and at the end of the studythey were sacrificed. Portions of tissues were collected and examinedfor human prostate pieces implanted earlier. PSA localizationdemonstrated presence of human prostate tissue. Thus, various parametersof drug effects can be readily established before undertaking in vivotreatment of prostatic tumors.

In Vitro Studies

[0066] Since human prostatic specific antigen and human prostatic acidphosphatase are not secreted by animal prostates, the invention wasinvestigated on human prostate tissues collected after surgery and humanprostate cell lines (such as DU-145, LNCaP) in cell culture and afterimplantation in nude mice. Human prostate tissues were investigated inorgan culture and after physical and chemical fixations and implantationin nude mice.

[0067]FIG. 1 illustrates the structure of the drug5-fluoro2-deoxyuridine and various derivatives that were evaluated aloneand after conjugation with rabbit anti-PSA and anti-PACP-IgGs. Thepurified immunoglobulin fraction of rabbit anti-sera were obtained fromDako (Dako Corp., Carpinteria, CA) and its purity was also evaluatedusing chemiluminescence immunoassay.

[0068] To determine whether the PSA IgG-drug-conjugate could be taken upby human prostate cells, small pieces of human prostates, collectedafter prostatectomies, were placed in organ culture media containingPSA-IgG conjugated with 5′fluoro-2′-deoxyuridine (5′-Fu-2′-d),5′-Fu-2′-d-biotin and/or 5′-fluoro-2′-deoxyuridine-5′-monophosphate(5′-Fu2′-d 2′-d 5′-mp) for 18 to 72 hours. Presence ofdrug-immunoconjugate in prostate specimens tissues was evaluated byimmunofluorescence, immunoperoxidase, and/or Dako kit localizationtechniques.

[0069]FIG. 2 shows a 225×magnification of randomly selected, small (1 to2 mm ) human prostate pieces that were incubated for 18 hours at 37° C.and 5% CO₂ in RPMI-1640 media without the immunoconjugate. The pieceswere washed in phosphate buffer saline (PBS), frozen in liquid nitrogen,embedded in OCT (an embedding media obtained from Lab Tek Products;Miles Lab. Inc., Naperville, Ill.), sectioned with a cryostat at 5 μm,fixed in 100% acetone and processed for the reaction products using theDako kit (Santa Barbara, Calif.). The section does not show any labelingbecause the PSA-IgG-drug immunoconjugate was not incorporated inprostate tissue.

[0070]FIG. 3 shows a 225× magnification of human prostate tissue thatwas incubated for 18 hours in RPMI-1640 media with an immunoconjugate;namely rabbit anti-PSA IgG-5′-FU2′d-biotin (1:20 dilution). Samples werewashed with PBS, frozen in liquid nitrogen, embedded in OCT, sectionedwith a cryostat at 5 μm, fixed with 100% acetone and processed for thelocalization of immunoconjugate using the Dako kit. The prostatic cellsshow products of the labeling reaction that are not present in thestromal tissues. Thus, immunostaining shows that the immunoconjugate isspecific for the epithelial cells.

[0071] To demonstrate the presence of PSA-IgG conjugated with 5′-Fu-2′-din prostate tumors, portions of prostate pieces were incubated in organculture media for 18 hours at 4° C., washed with PBS, sectioned with acryostat, fixed with 100% acetone and incubated with goat anti-rabbitIgG conjugated with fluorescein isothiocyanate (FITC) for 1 hour.Sections were examined for fluorescence using a Zeiss epifluorescencemicroscope and a Zeiss blue barrier filter. FIG. 4 illustrates (225×magnification) the presence of PSA-IgG-drug-immunoconjugate inepithelial cells of human prostate, but not in the stromal tissuesbetween the glands. Immunofluorescence techniques provided resultscomparable to those in FIG. 3. These results confirm the specificity ofthe immunoconjugate for the prostatic epithelial cells.

[0072] That both PSA-IgG and 5′-Fu-2′-d-5′-mp reach the same groups ofprostate cells is demonstrated by a double immunofluorescence technique.For this purpose, PSA-IgG was conjugated with rhodamine (which producesred fluorescence) and 5′-Fu-2′-d-5′-mp was conjugated with fluoresceinisothiocyanate (FITC) (which produces blue fluorescence). Theseseparately labeled products were then conjugated together producingPSA-IgG-rhodamine-5′-Fu-2′-d-5′-mp-FITC (PRFF) immunoconjugate. Cryostatsections were then incubated with PRFF for 1 or 18 hours. Human prostatesection showing a Gleason histologic score 5 cancerous prostate tumorwas examined using a Zeiss epifluorescence microscope and Zeiss greenand blue excitation and barrier filters, respectively. FIG. 5illustrates (225× magnification) that localization of red coloredrhodamine occurred in epithelial cells, and not in prostatic stromaltissues which do not produce prostatic specific antigen.

[0073]FIG. 6 shows the result (magnification ×225) when a blue filterwas used to visualize the presence of drug in the same group ofprostatic tumor cells. In other words, the double immunofluorescencetechnique showed that the drug was indeed conjugated to PSA-IgG and itreached the target cells, i.e., prostatic epithelial cells.

[0074]FIG. 7 demonstrates the presence of stromal tissues betweenprostatic glands shown in FIGS. 5 and 6. The prostate section used forrhodamine and fluorescein localization was photographed with a Zeissmicroscope equipped with phase contrast. This micrograph (magnification×225) demonstrates the presence of stromal cells between prostaticglands, but that localization of the immunoconjugate occurred only inthe epithelial cells.

[0075] To demonstrate that rhodamine and FITC-labeled immunoconjugatewas specific for human prostatic epithelial cells, human kidney,bladder, and colon cryostat sections were incubated with PRFFimmunoconjugate. FIG. 8 (magnification ×225) illustrates the absence ofrhodamine or FITC localization in the human kidney. Likewise, bladderand colon sections did not show localization of PRFF immunoconjugate(not illustrated). This study demonstrated that PSA-IgG-drug conjugatetargeted only prostatic epithelial cells containing prostatic specificantigen. Since the antigen was not present in kidney, bladder, or colon,no localization occurred.

[0076] To demonstrate further that PSA-IgG functions as a carrierprotein for 5′-Fu-2′-d, the drug was labeled with FITC alone. Theconjugate did not contain PSA-IgG. Human prostate sections wereincubated for 18 hours with 5′-Fu2′-FITC labeled drug and examined witha Zeiss epifluorescence microscope. FIG. 9 (magnification ×225) showsthe absence of fluorescence in prostatic epithelial or stromal cells,indicating that the drug alone was not specific for prostate cells andthat prostatic specific antigen was needed for carrying and binding thedrug to epithelial cells.

[0077] When human prostate sections were incubated with normal rabbitserum without the immunoconjugate PSA-IgG-drug conjugate, followed bytreatment with goat anti-rabbit conjugated with FITC, i.e. a negativecontrol experiment, no fluorescence was observed in prostatic epithelialcells, as can be seen in FIG. 10 (magnification ×225), demonstrating thespecificity of PSA antibody IgG.

[0078] The results shown in FIGS. 2-10 demonstrate that PSA-IgG-drugconjugate binds only to prostatic epithelial cells possessing prostaticspecific antigen and it does not bind to any other type of tissuesexamined. Furthermore, drug alone did not bind to prostatic cells. Thenegative control experiment demonstrated specificity of the PSA-IgGantibody. Taken together, these figures have shown specificity ofPSA-IgG-drug immunoconjugate for neoplastic prostate epithelial cells.Differential immunofluorescence has demonstrated that PSA-IgG functionsas a carrier molecule for the chemotherapeutic drug into prostate cells.

[0079] The following experiment demonstrates presence of PSA moleculesin benign prostatic hyperplasia (BPH).

[0080] A cryostat section of human benign prostatic hyperplasia wasincubated for 18 hours with immunoconjugate PRFF. In this section, thepresence of rhodamine in epithelial cells is shown using Zeissepifluorescence microscope and a red barrier filter. Again, as shown inFIG. 11 (magnification ×225) rhodamine labeling was present only inepithelial cells and not in stromal tissue cells.

[0081] Upon analyzing the above section using a blue filter, theobserved fluorescence (FIG. 12-225× magnification) demonstratedlocalization of 5′-Fu-2′-d-5′-mp labeled with FITC in the above group ofprostatic epithelial cells.

[0082]FIG. 13 illustrates the above section as photographed with a Zeissmicroscope equipped with phase contrast (magnification ×225). Thissection shows details of glandular epithelium and stromal tissues shownin FIGS. 11 and 12. This micrograph further demonstrates that bothrhodamine and fluorescein labeling occurred only in epithelial cells asexpected.

[0083] The above shows that PSA-IgG functions as a carrier protein forthe conjugated chemotherapeutic agents. The immunoconjugate targetedprostatic epithelial cells and their tumors selectively . Organ culturestudy showed this immunoconjugate did reach the glandular cells andbound the epithelial cells. Thus, the immunoconjugate is highly specificfor prostatic tumors.

[0084] In addition to conjugating PSA-IgG with derivatives of5′-fluorouracil, we conjugated anti-human prostatic acid phosphatase IgGwith 5′-fluoro-2′-deoxy-5′-monophosphate. This resulted in the formationof the immunoconjugate, PAcP-IgG-5′-Fu′2′-d5′-mp. This immunoconjugate,much like the PSA-IgG-drug immunoconjugate, was labeled with a varietyof markers (such as rhodamine, fluorescein) and evaluated on humanprostate tissue samples obtained after prostatectomy. Human prostaticacid phosphatase is another highly specific marker for human prostate,and the PAcP-IgG-drug immunoconjugate was tested to determine whetherthe latter immunoconjugate would behave similarly to PSA-IgG-drugimmunoconjugate. Much as the PSA-IgG-drug immunoconjugate, thePAcP-IgG-drug immunoconjugate was highly specific for human prostatetumors and it did not bind to epithelial cells of human colon, bladder,kidney, or lung.

[0085] In addition, several types of chemotherapeutic agents have beenused to treat human prostate and other solid organ tumors (Yogoda, A.,and Petrylak, D., Cancer 71: 1098-109, 1993; Rangel, C., Matzkin, H.,and Soloway, M. S., Urology, 39:577-582, 1992; Tritton, T. R., and Yee,G., Sci., 217:248-250, 1982.). Thus, the present invention was appliedto another bioactive agent, doxorubicin (an anthracycline). There arenumerous derivatives of anthracyclines, some of which are involved incytotoxicity and DNA synthesis inhibition. Doxorubicin causescytotoxicity without entering cells (unlike the fluorouracilderivatives) and it also inhibits DNA synthesis and mitosis. Doxorubicinhas been used in the treatment of prostate cancer (Yogoda, A., andPetrylak, D., Cancer 71: 1098-109, 1993; Rangel, C., Matzkin, H., andSoloway, M. S., Urology, 39:577-582, 1992). These treatments requiredutilization of very high dosages of drugs and showed variable results.Doxorubicin alone is not specific for prostate cancer and affects manyother organs in patients.

[0086] Doxorubicin was conjugated with PSA-IgG and the immunoconjugatePSA-IgG-doxorubicin resulted. We also conjugated doxorubicin alone withfluorescein. Immunoconjugate prepared using PSA-IgG labeled withrhodamine and doxorubicin with fluorescein was also evaluated on humanprostate samples. Using single and double conjugated doxorubicin withmarkers, it was shown that the immunoconjugate was specific for humanprostate as it did not localize in human colon, kidney, bladder, andlung. Our study showed that PSA-IgG-doxorubicin immunoconjugate wascarried to prostatic epithelial cells only when PSA-IgG molecules werepresent. The drug alone did not specifically localize in prostaticepithelial cells. This immunoconjugate did not bind to epithelial orstromal cells.

[0087] The present immunoconjugates have the major advantages in thetreatment of prostatic diseases (such as prostate cancer, BPH,prostatitis) in contrast to unconjugated chemotherapeutic bioactiveagents or toxins. Because of the prostate epithelial cell specificity,the immunoconjugates could be the drug of choice for the treatment ofprostatic tumors and prostatic tumor metastases. These immunoconjugatescould be used in lower therapeutic doses than any conventionaltreatments currently being used. Furthermore, the immunoconjugatetreatment would specifically target prostate tumors cells.

Nude Mice Study

[0088] A study was designed to determine the effects ofanti-PSA-IgG-drug complex (immunoconjugate) administered into humanprostate cell (LNCaP) tumors grown subcutaneously in nude mice. Thisexperiment was designed to study the cytotoxic effects of the IgG-drugconjugate by administering the agent in the MATRIGEL supporting thetumor cells. MATRIGEL is a solubilized tissue basement membranepreparation extracted from the Engelbreth-Holm-Swarm (EHS) mouse tumor,available from Becton-Dickinson (Collaborative Research Inc, BedfordMass. MATRIGEL is made predominantly of laminin with type IV collagen,heparan sulfate proteoglycans, etc., polymerized under physiologicalconditions to produce a reconstituted biologically active matrixmaterial that serves as an in vivo attachment matrix which also promotescell differentiation in normal and transformed cells. Basement membranesare thin extracellular membranes underlying epithelial cells andseparation them from connective tissue. Basement membranes (such asMATRIGEL) allow growth and differentiation into specialized tissues andmoderate and control cellular behavior. Five groups of mice wereevaluated in this study.

[0089] Group 1. Mice were injected subcutaneously with Matrigel alone(control preparation), no tumor cells and phosphate buffer saline ascontrol media. Analysis of these injection sites revealed that theMatrigel did not contain tumor cells since no tumor cells were injected.The morphology of the Matrigel is illustrated in FIG. 14).

[0090] Group 2. Mice were injected subcutaneously with Matrigelcontaining 10⁶ LNCaP tumor cells and phosphate buffer saline as controlmedia. Analysis of the animals revealed that tumor cells did grow in theMatrigel and formed numerous colonies (FIG. 15).

[0091] Group 3. Mice were injected subcutaneously with Matrigelcontaining 106 LNCaP tumor cells with the immunoconjugate and phosphatebuffer saline mixed in with Matrigel and cells at the time of injection.Analysis of these mice (FIG. 16) indicated that tumor cells present inthe Matrigel were unable to form large colonies in contrast to thecolonies shown in FIG. 15.

[0092] Group 4. Mice were injected subcutaneously with Matrigelcontaining 106 LNCaP cells and anti-PSA IgG alone (antibody control).Analysis of this group of mice showed that tumor cells formed colonies,but the number of colonies was fewer and smaller than in the Group 2mice. The pattern of colony formation was similar to those of the Group2 mice (FIG. 17).

[0093] Group 5. Mice were injected subcutaneously with Matrigelcontaining 106 LNCaP cells. These tumors were allowed to grow for 21days, at which time they received two sequential injections of anti-PSAIgG-drug conjugate. These mice were followed for changes in tumor sizeand sacrificed 30 days after the beginning of injection treatments. Inthe group of mice treated with the immunoconjugate, the number of tumorcell colonies was fewer than in any of the groups evaluated earlier(FIG. 18).

[0094] The study on nude mice shows that the immunoconjugate was moreeffective in reducing the number of tumor colonies than was the antibodyalone (group 4). It is interesting to note that very early treatmentwith the immunoconjugate prevented formation of large colonies. Itshould be noted that the active agent was administered in Groups 3 and 5in an amount of about 65 μg/g, suggesting that similar results could beobtained for a range of at least about 50-100 μg/g. Ordinarily, it wouldbe expected that a dosage of about 500 μg/g would be necessary toachieve the results seen in Group 5.

[0095] In our study, we found that measuring tumors while still growingin nude mice is not as accurate, especially when tumors were small, asthe measurement and weighing them after excision. Since the in situmeasurement of tumors was not suitable, we evaluated the immunoconjugateeffects on tumors by localization of markers for cell proliferation(such as proliferation cell nuclear antigen:PCNA) and cell death(nuclear DNA fragmentation). Preliminary evaluation of our dataindicated that in immunoconjugate treated mice there were fewerproliferating cells than in the untreated mice. Furthermore, the treatedtumors showed increased cell death over the untreated group. These twomethods could also be evaluated by establishing immunoconjugatetreatment for human prostate tumors implanted in nude mice as well asfor human prostate tumors in organ culture.

[0096] With the advent of newer techniques (such as ultrasonic imaging,PSA in serum), many incidental and early stage prostate cancers arebeing diagnosed. Some of these tumors develop metastatic disease, whileothers remain dormant. In some cases, watchful waiting treatment isgiven while others may receive prostatectomy and/or hormonal treatment.Prostatectomy has high morbidity, altered life style, and evenmortality. In the early stage and incidental prostate cancers, theimmunoconjugates could be used as well.

[0097] For example, early stage tumors, which are often confined to theprostate gland, could be injected with the immunoconjugate. Theimmunoconjugate would be given in therapeutic dosages and directly intointraprostatic tumor sites via the transurethral route. In this casedirect injection may prove to be very effective since it would bind toprostatic epithelial cells tumor, inhibit DNA synthesis, and producecytotoxic effects. Infusion of the immunoconjugate in the prostate hasthe advantage of draining the immunoconjugate via prostatic lymphaticsand small venules. This drainage system would undoubtedly producecytotoxic effects on prostate tumor cells if they are present inregional pelvic lymph nodes as well as in the pelvic bone areas.

[0098] Another problem with the PSA or PAcP antibody-basedimmunoconjugate is that of the immunoconjugate binding to serum PSA orPAcP, since these may be increased in cancer or BPH patients. It ispossible that substantially higher dosages of immunoconjugates will berequired in human patients. However, there are methods available toovercome this drawback. For example:

[0099] a) Initially, patients will be treated with humanized,unconjugated antibody which would bind serum PSA (or PAcP) and thiscould be followed by immunoconjugate treatment. This approach wouldallow the immunoconjugate to reach the target primary tumor as well asmetastatic prostate tumor cells, since the unconjugated antibody wouldhave bound the serum PSA (or PAcP) with the unconjugated, humanizedantibody.

[0100] b) Another approach to reduce binding of immunoconjugate to serumPSA (or PACP) would be to treat prostate cancer patients with thecurrent conventional hormone and/or chemotherapeutic treatments for ashort period of time. This would reduce serum PSA (or PAcP) to a verylow level. The immunoconjugate treatment then could be utilized fortargeting prostatic tumor cells. Similar approaches could be used toovercome similar problems in the treatment of other conditions and othertissues and organs.

[0101] The invention has been described with reference to variousspecific and preferred embodiments and techniques. However, it should beunderstood that many variations and modifications may be made whileremaining within the spirit and scope of the invention.

[0102] All publications and patent applications in this specificationare indicative of the level of ordinary skill in the art to which thisinvention pertains. All publications and patent applications are hereinincorporated by reference to the same extent as if each individualpublication or patent application was specifically and individuallyindicated by reference.

[0103] The above specification, examples and data provide a completedescription of the manufacture and use of the composition of theinvention. Since many embodiments of the invention can be made withoutdeparting from the spirit and scope of the invention, the inventionresides in the claims hereinafter appended.

What is claimed is:
 1. An immunoconjugate that recognizes a substanceunique to an organ, comprising an antibody or derivative or fragmentthereof for said substance, linked to a bioactive agent.
 2. Theconjugate of claim 1 wherein the organ is a prostate gland.
 3. Theconjugate of claim 2 wherein the prostate is cancerous.
 4. The conjugateof claim 2 wherein the prostate is a human prostate.
 5. Theimmunoconjugate of claim 1 wherein the antibody is an IgG.
 6. Theconjugate of claim 1 wherein the antibody is an antibody fragment. 7.The conjugate of claim 1 wherein the bioactive agent is selected fromthe group consisting of a toxin, an enzyme inhibitor, a receptorantagonist, a molecule that interferes with macromolecular interactions,an antimetabolite, and a chemotherapeutic agent.
 8. The conjugate ofclaim 1 wherein the bioactive agent interrupts DNA or RNA synthesis. 9.The conjugate of claim 1, wherein the antibody is a polyclonal antibody.10. A method of delivering a bioactive substance to an organ in ananimal, comprising administering to the animal an immunoconjugate whichcomprises an antibody or derivative or fragment thereof for a substancewhich is unique to the organ, and a bioactive agent linked to theantibody or derivative or fragment thereof.
 11. The method of claim 10wherein the animal is human.
 12. The method of claim 10 wherein theorgan is afflicted with a solid organ tumor and the bioactive agent hasactivity against the tumor cells.
 13. The method of claim 10, whereinthe antibody is a polyclonal antibody.
 14. The method of claim 10,wherein the organ is the prostate.
 15. The method of claim 14, whereinthe prostate is afflicted with adenocarcinoma of the prostate, benignprostatic hypertrophy or prostatitis.